Iron Insufficiency Compromises Motor Neurons and Their Mitochondrial Function in Irp2-Null Mice

Genetic ablation of Iron Regulatory Protein 2 (Irp2, Ireb2), which post-transcriptionally regulates iron metabolism genes, causes a gait disorder in mice that progresses to hind-limb paralysis. Here we have demonstrated that misregulation of iron metabolism from loss of Irp2 causes lower motor neuronal degeneration with significant spinal cord axonopathy. Mitochondria in the lumbar spinal cord showed significantly decreased Complex I and II activities, and abnormal morphology. Lower motor neurons appeared to be the most adversely affected neurons, and we show that functional iron starvation due to misregulation of iron import and storage proteins, including transferrin receptor 1 and ferritin, may have a causal role in disease. We demonstrated that two therapeutic approaches were beneficial for motor neuron survival. First, we activated a homologous protein, IRP1, by oral Tempol treatment and found that axons were partially spared from degeneration. Secondly, we genetically decreased expression of the iron storage protein, ferritin, to diminish functional iron starvation. These data suggest that functional iron deficiency may constitute a previously unrecognized molecular basis for degeneration of motor neurons in mice

Functional Expression of AQP3 in Human Skin Epidermis and Reconstructed Epidermis

The purpose of this study was to examine the presence of aquaporin water channels in human skin and to assess their functional role. On western blots of human epidermis obtained from plastic surgery, a strong signal was obtained with polyclonal anti-aquaporin-3 antibodies. By indirect immunofluorescence on 5 µm cryosections, anti-aquaporin-3 antibodies strongly stained keratinocyte plasma membranes in human epidermis, whereas no staining was observed in the dermis or the stratum corneum or when anti-aquaporin-3 antibodies were preabsorbed with the peptide used for immunization. Similarly, a strong signal with anti-aquaporin-3 antibodies was observed in keratinocyte plasma membranes of reconstructed human epidermis in culture at the air–liquid interface for up to 3 wk. The keratinocyte plasma membrane localization of aquaporin-3 was confirmed at the electron microscope level in prickle cells. In addition an intracellular localization of aquaporin-3 was also detected in epidermis basal cells. Osmotically induced transepidermal water permeability was measured on stripped human skin and on reconstructed epidermis. Water transport across both stripped human skin and 2–3 wk reconstructed epidermis was comparable, inhibited by > 50% by 1 mM HgCl2 and fully inhibited by acid pH. By stopped-flow light scattering, keratinocyte plasma membranes, where aquaporin-3 is localized, exhibited a high, pH-sensitive, water permeability. Although human skin is highly impermeable to water, this is primarily accounted for by the stratum corneum, where a steep water content gradient was demonstrated. In contrast, the water content of viable strata of the epidermis is remarkably constant. Our results suggest that the human epidermis, below the stratum corneum, exhibits a high, aquaporin-3-mediated, water permeability. We propose that the role of aquaporin-3 is to water-clamp viable layers of the epidermis in order to improve the hydration of the epidermis below the stratum corneum

Interaction between the Triglyceride Lipase ATGL and the Arf1 Activator GBF1

The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets

Electron Tomography of the Contact between T Cells and SIV/HIV-1: Implications for Viral Entry

The envelope glycoproteins of primate lentiviruses, including human and simian immunodeficiency viruses (HIV and SIV), are heterodimers of a transmembrane glycoprotein (usually gp41), and a surface glycoprotein (gp120), which binds CD4 on target cells to initiate viral entry. We have used electron tomography to determine the three-dimensional architectures of purified SIV virions in isolation and in contact with CD4+ target cells. The trimeric viral envelope glycoprotein surface spikes are heterogeneous in appearance and typically ∼120 Å long and ∼120 Å wide at the distal end. Docking of SIV or HIV-1 on the T cell surface occurs via a neck-shaped contact region that is ∼400 Å wide and consistently consists of a closely spaced cluster of five to seven rod-shaped features, each ∼100 Å long and ∼100 Å wide. This distinctive structure is not observed when viruses are incubated with T lymphocytes in the presence of anti-CD4 antibodies, the CCR5 antagonist TAK779, or the peptide entry inhibitor SIVmac251 C34. For virions bound to cells, few trimers were observed away from this cluster at the virion–cell interface, even in cases where virus preparations showing as many as 70 envelope glycoprotein trimers per virus particle were used. This contact zone, which we term the “entry claw”, provides a spatial context to understand the molecular mechanisms of viral entry. Determination of the molecular composition and structure of the entry claw may facilitate the identification of improved drugs for the inhibition of HIV-1 entry

Three-Dimensional Aberration-Corrected Scanning Transmission Electron Microscopy for Biology

Recent instrumental developments have enabled greatly improved resolution of scanning transmission electron microscopes (STEM) through aberration correction. An additional and previously unanticipated advantage of aberration correction is the greatly improved depth sensitivity that has led to the reconstruction of a three-dimensional (3D) image from a focal series. In this chapter the potential of aberration-corrected 3D STEM to provide major improvements in the imaging capabilities for biological samples will be discussed. This chapter contains a brief overview ofthe various high-resolution 3D imaging techniques, a historical perspective of the development of STEM, first estimates of the dose-limited axial and lateral resolution on biological samples and initial experiments on stained thin sections

A role for actin arcs in the leading-edge advance of migrating cells

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion. © 2011 Macmillan Publishers Limited. All rights reserved

Hologenome analysis of two marine sponges with different microbiomes

Background: Sponges (Porifera) harbor distinct microbial consortia within their mesohyl interior. We herein analysed the hologenomes of Stylissa carteri and Xestospongia testudinaria, which notably differ in their microbiome content. Results: Our analysis revealed that S. carteri has an expanded repertoire of immunological domains, specifically Scavenger Receptor Cysteine-Rich (SRCR) like domains, compared to X. testudinaria. On the microbial side, metatranscriptome analyses revealed an overrepresentation of potential symbiosis-related domains in X. testudinaria. Conclusions: Our findings provide genomic insights into the molecular mechanisms underlying host-symbiont coevolution and may serve as a roadmap for future hologenome analyses

Phoenix Is Required for Mechanosensory Hair Cell Regeneration in the Zebrafish Lateral Line

In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration

Multilayered Mechanism of CD4 Downregulation by HIV-1 Vpu Involving Distinct ER Retention and ERAD Targeting Steps

A key function of the Vpu protein of HIV-1 is the targeting of newly-synthesized CD4 for proteasomal degradation. This function has been proposed to occur by a mechanism that is fundamentally distinct from the cellular ER-associated degradation (ERAD) pathway. However, using a combination of genetic, biochemical and morphological methodologies, we find that CD4 degradation induced by Vpu is dependent on a key component of the ERAD machinery, the VCP-UFD1L-NPL4 complex, as well as on SCFβ-TrCP-dependent ubiquitination of the CD4 cytosolic tail on lysine and serine/threonine residues. When degradation of CD4 is blocked by either inactivation of the VCP-UFD1L-NPL4 complex or prevention of CD4 ubiquitination, Vpu still retains the bulk of CD4 in the ER mainly through transmembrane domain interactions. Addition of a strong ER export signal from the VSV-G protein overrides this retention. Thus, Vpu exerts two distinct activities in the process of downregulating CD4: ER retention followed by targeting to late stages of ERAD. The multiple levels at which Vpu engages these cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1

Unravelling surface and interfacial structures of a metal–organic framework by transmission electron microscopy

Metal-organic frameworks (MOFs) are crystalline porous materials with designable topology, porosity and functionality, having promising applications in gas storage and separation, ion conduction and catalysis. It is challenging to observe MOFs with transmission electron microscopy (TEM) due to the extreme instability of MOFs upon electron beam irradiation. Here, we use a direct-detection electron-counting camera to acquire TEM images of the MOF ZIF-8 with an ultralow dose of 4.1 electrons per square ångström to retain the structural integrity. The obtained image involves structural information transferred up to 2.1 Å, allowing the resolution of individual atomic columns of Zn and organic linkers in the framework. Furthermore, TEM reveals important local structural features of ZIF-8 crystals that cannot be identified by diffraction techniques, including armchair-type surface terminations and coherent interfaces between assembled crystals. These observations allow us to understand how ZIF-8 crystals self-assemble and the subsequent influence of interfacial cavities on mass transport of guest molecules